The control of cell proliferation and differentiation in the normal breast and in breast cancer involves complex actions and interactions of steroid hormones (in particular estrogen and progesterone), peptide hormones and growth factors (1, 2). How these agents act at critical control points within the cell cycle to influence progression through the cycle or exit to enter a pathway of differentiation is only partially understood (3–5).
Progestins are responsible for mammary gland lobuloalveolar development during pregnancy (6), although there is evidence for a more predominant role for estrogens than progestins in stimulating epithelial cell proliferation in the normal premenopausal breast (7, 8). Progestins both stimulate and inhibit breast cancer epithelial cell proliferation in vitro but the predominant effect is growth inhibition probably via induction of differentiation (3, 4, 7, 9). Progestin action is mediated primarily through the progesterone receptor (PR), which acts as a transcriptional transactivator for a largely undefined set of progestin-responsive genes which may, in turn, transcriptionally or post-transcriptionally influence additional genes or gene products.
Only a limited number of genes have been implicated in progestin action on cell proliferation. Previous studies by the present inventors have identified c-myc and cyclin D1 as major downstream targets of progestin-stimulated cell cycle progression in human breast cancer cells (3, 10) while the delayed growth inhibitory effects of progestins involve decreases in cyclin D1 and E gene expression (4, 9). While progestin effects on c-myc gene expression are rapid and occur within minutes, effects on cyclin expression begin several hours later, pointing to the presence of undefined earlier events.
Since progestin action is complex and is likely to involve multiple genes, many of which are currently unknown, the differential display RT-PCR technique (DD-PCR) (11) was adopted to identify target genes in cultured human breast cancer cells. The utility of this approach has been previously demonstrated by the cloning of PRG1, a gene having significant homology with isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (12). Using the same technique, a novel progestin-regulated gene, EDD (designated DD5 in the applicant's Australian Provisonal patent application No. PO6334), has been identified.
Based on amino acid sequence similarity, EDD appears to be a human homologue of the Drosophila tumor suppressor gene hyperplastic discs (hyd) (13). Although the function of the HYD protein is unknown, significant homology exists between its carboxyl terminus and those of human E6-AP and a number of proteins identified through database searches (14). These HECT domain family proteins function as ubiquitin-protein ligases (E3 enzymes) (14–16), playing a role in the ubiquitination cascade that targets specific substrate proteins for proteolysis. Notably, the protein encoded by EDD has a carboxy-terminal HECT domain containing a cysteine residue that covalently binds ubiquitin. This amino acid is conserved in all known HECT domain-containing E3 enzymes and is involved in the transfer of ubiquitin. It is therefore proposed that the EDD gene represents a novel human tumour suppressor gene encoding a ubiquitin-protein ligase.